The formation of Islets of Langerhans from progenitor cells during pancreas development requires the coordinated activation of the Delta/Notch signaling pathway. This pathway, although it has been implicated in both maintaining multipotency and supporting cell fate decisions of pancreatic progenitor cells, is poorly understood. In order to better understand these and other events that occur during pancreas development, it is important to be able to easily examine the effects, within the developing mouse pancreas, of both gain-of-function and loss-of-function mutations. When possible, it is also useful for these mutations to be conditionally active or inactive. Thus, this project is focused on both the use and further development of cassette exchange technologies in mouse ES cells. This strategy accelerates the production of highly defined gene mutations in ES cells, which can then be used either to make new mice or as tools for learning how to direct the differentiation of ES cells towards a pancreatic or beta cell fate. The reagents that will be developed, as well as the experiments that will be performed, promise to accelerate progress towards an understanding of the molecular events that give rise to endocrine cells within the pancreas. In Aim 1 we will explore the growth and differentiation capacity of pancreatic progenitor cells using mice in which yellow fluorescent protein is expressed under control of the Ptf1a gene locus. For Aim 2 we will explore alterations in Delta/Notch signaling by expressing three different fringe genes under control of the Ptf1a gene locus. In Aim 3 we will assess the effects of a global knock-out of Insm1 while also generating a new loxed cassette acceptor allele using an improved strategy. For Aim 4 we will generate and test new mouse ES cell lines that will facilitate learning how to direct the differentiation of these cells towards a pancreatic beta cell fate. Lastly, in Aim 5 we will establish a new cassette exchange system that makes use of phiC31 integrase to allow rapid insertion of genes into the ROSA26 locus and their subsequent induction in a site- and temporal-specific manner by Cre.